The Fc-gamma-receptor IIIb (FcgRIIIB) genes that encode neutrophil-specific antigens NA1 and NA2 differ at five nucleotides, four that result in amino acid differences among the two alleles. The role of each of these amino acid differences in antigen expression is not known. People with FcgRIIIB genes that differ from the NA1-FcgRIIIB and NA2-FcgRIIIB by a single nucleotide have been described. The purpose of this study was to compare the expression of NA1 and NA2 on granulocytes among people with variant FcgRIIIB genes and healthy blood donors. Reactions of NA1- and NA2-specific monoclonal and alloantibodies with granulocytes were assessed using flow cytometry in 74 healthy blood donors and six people with known variant FcgRIIIB genes. The granulocytes were tested with one NA1-specific monoclonal antibody, one NA2-specific monoclonal antibody, 4 NA1-specific alloantibodies and 4 NA2 specific alloantibodies. Analysis of the granulocytes from people with variant NA genotypes revealed that single base substitutions in FcgRIIIB at 141 and in FcgRIIIB at 349 are important in the expression of NA1 and single base substitutions in FcgRIIIB at 227 and 277 are important in the expression of the NA2. Among blood donors, age, gender, or race did not affect the expression of NA1 and NA2. The NA2-specific monoclonal antibody reacted more intensely with granulocytes from NA2-homozygous cells than heterozygous cells but this was not true for NA2-specific alloantibodies. There was no difference in the mean reactions of the NA1-specific monoclonal and alloantibodies among cells from NA1-homozygous and NA-heterozygous donors. The intensity of reactions of both the NA1-specific monoclonal and alloantibodies were strongly correlated on homozygous cells but not heterozygous cells as were the reactions of the NA2-specific monoclonal and alloantibodies. In fact, granulocytes from seven healthy blood donors who phenotyped as NA-heterozygous with the monoclonal antibodies were phenotyped as NA2-homozygous with the alloantibodies. It is known that variations in FcgRIIIB are common in African Americans, but five of these donors were Caucasian. These results suggest that variations in FcgRIIIB may be common in both Caucasians and African Americans. The expression of NA2 is dependent on the polymorphisms in FcgRIIIB 227 and 277 both of which are involved in an FcgRIIIB N-glycosylation site. Polymorphisms in FcgRIIIB at 141 and 349 appear more important to the expression of NA1.